Biochimica et Biophysica Acta, 1023 (1990) 223-229
223
Elsevier BBAMEM 74798
Single-channel currents activated by low intracellular pH in cultured hippocampal neurons of rat Fabio Franciolini Istituto Biologia CeUulare UnioersiMdi Perugia, Perugia (Italy) and Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL (U.S.A.)
(Received 11 July 1989) (Revised manuscript received1 November 1989)
Key words: Single-channelcurrent; Patch damp; Ion channel; pH; (Rat neuron)
Patch damp technique was applied to the plasma membrane of cultured hippucampai neurons of rat. Elementary currents of a cation-selective channel were elicited by low intraceilular pH (pH i 3.5-4.5). Channel activity starts with 1-2 min delay from the application of low pH i, and persists upon restoration of physiological pH conditions. The channel has a conductance of approx. 110 pS in symmetrical 300 mM NaCI, and is strongly selective for cations over anions. The channel is active over the whole voltage range tested (from + 75 mV to - 7 5 mV). Mean open time is function of voltage, increasing with depolarization. Low pH applied extracellularly did not activate the channel.
Introduction The effects of high proton concentration on ionic channel activity have been extensively studied. Reports broadly fall into three categories. (1) Inhibition of channel activity. Low (external or internal) p H has been shown to block conduction in the squid giant axon [1], and to decrease conductance or activity of Na ÷ channels [2-7], K ÷ (delayed rectifier, inward rectifier, Ca2+-activated) channels [4,8-13], Ca 2÷ channels [14-20], C1- channels [21-24], and acetylcholine receptor [25]. A few studies have also been done on the effect of intracellular p H on the inactivation of the Na ÷ channel. Two apparently conflicting reports are mentioned here. Na + current inactivation is removed or greatly reduced by high p H i in squid giant axon [26], but by low p H i in frog skeletal muscle [27]. (2) Production of H ÷ currents. H ÷ currents have been recently described by several authors in different preparations. Mozhayeva and Naumov [28] report a H ÷ current at p H lower than 4 that permeates through Na ÷ channels. In snail neurons, H + currents have been first reported by Thomas and Meech [29], and described in more detail by Byerly et al. [30]. The latter paper shows that H + currents go through specific channels which are very selective for H +.
Correspondence: F. Franciolini, Istituto BiologiaCeUulare,via Pascoli 1, 06100 Perugia, Italy.
(3) Transformation of one channel type into another. Lux and co-workers [31,32] found that low external p H (pHo) activates a transient N a ÷ current. Two major observations induced the authors to conclude that the Na ÷ current activated by low p H o flows through a transformed state of a Ca 2÷ channel. First, the voltagegated Ca 2÷ current disappeared rapidly during the activation of proton-induced Na ÷ current. Ca 2÷ current reappeared, however, while Na + current inactivated, upon restoration of physiological p H conditions. This 'temporal exclusiveness' of the two currents suggested that both Na + and Ca 2+ currents use the same Ca 2+ channel in two selectively distinct states. Additional evidence in support of this view was the finding that the proton-activated Na + current was blocked by organic (diltiazem, verapamil) and inorganic (Ni and Cd) Ca 2÷ channel blockers at concentrations similar to those required to block the Ca 2÷ currents. For completeness, one report has also appeared on proton modulation of C1- channels reconstituted from Torpedo electroplax [33], which shows that lowering pH increases the open probability of the channel, whereas rising it has the opposite effect. A change in p K of certain titratable groups upon opening of the channel is proposed to explain the pH-modulation of the channel gating. This paper reports on the activation of a cationic channel by low internal pH. The results are discussed in the light of previous reports, to try to determine to which category the effects reported can be ascribed, and
0005-2736/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
224 what possible mechanisms underly the proton-activated cationic channel in cultured hippocampal neurons of rat.
maintaining that value for tens of seconds to minutes, or by stimulating the membrane with a staircase pattern of voltage steps that started with positive steps to activate the channels, and applied over a period of time during which sufficient activity was sustained (for details, cf. Ref. 36). Current amplitude histograms of single-channel recordings were constructed for both stimulation protocols, and used to measure single-channel current and channel open probability. Single-channel current was measured as interpeak distance, and channel open probability calculated from the integral areas of the amplitude histogram peaks. The solutions used contained either 300 or 1200 mM of bulk salt (NaC1 or KC1), 1 mM EGTA, and 2 mM of pH buffer. Solutions where the pH was adjusted to 7.2 were buffered with Mops, and those adjusted to pH 3.5-4.5 were buffered with citric acid. In the text, these solutions are referred to by specifying the concentration and species of the bulk salt, and the pH; for instance, 300 NaC1 (pH 4.2). Effective change of test solutions at the cytoplasmic side of the patch in the inside-out configuration was obtained with a perfusion system similar to that described by Franciolini and Nonner [36]. Experiments were carried out at 25-28 o C.
Methods
Experiments were carried out on hippocampal neurons dissected from 18-day-old rat embryos, and plated on collagene/polylysine-coated culture dishes containing basic tissue culture medium (N5) supplemented with a fraction of horse serum [34]. Neurons were kept in culture at 34°C in an atmosphere containing 5% CO 2 for 1-3 weeks before the experiment. The patch clamp method [35] was used to record single-channel currents in the inside-out configuration, that is, with the cytoplasmic side of the membrane exposed to the perfusing solution. A homemade patch clamp amplifier was used for voltage clamping and current amplification. Single-channel currents were filtered at 0.5 kHz ( - 3dB), digitized at 400 #s sampling rate, and analyzed with a laboratory computer system (Nova 4, Data General). Single-channel records were taken in either steady-state conditions attained by stepping the membrane voltage to the desired level, and A
12OO NaCI,
pH Z 2 / / 3 0 0
NaCI,
pH 7.2
closed
B
12OO N a C I ,
pH 3 . 9 / / 3 0 0
NaCI, pH 7.2
/
~| ..I
LLI.t_.I _h
_]_|j~
pA : ms :
25 1560
Fig. 1. Activation of the cationic channel by low pH i. Single-channel recordings from an excised inside-out patch of hippocampal neuron of rat were taken in steady-state conditions at holding potential of 0 mV. The traces are consecutive 1560-ms segments interrupted by 20-ms blank intervals due to data aquisition. Pipette solution was 300 m M NaC1 (pH 7.2) and perfusing solution was 1200 m M NaC1 (pH 7.2) for the first two control traces (A), and 1200 m M NaC1 (pH 3.9) for the following traces (B). The traces shown in (B) were taken 80 s after solution change. Calibration: solid lines enclosing single channel recordings span 25 pA.
225 Results
In an attempt to elucidate the permeation mechanism of the background C1- channel in hippocampal neurons of rat, a series of experiments were carried out at low intracellular pH, ranging from 7.0 to 4.0. Low pH i was found to abolish the activity of the background C1- channel (Franciolini and Nonner, unpublished data). Often, however, the activation of a single-channel cationic current with distinctive conductance and kinetics was observed, concurrent with the depression of C1current. This is illustrated in Fig. 1 which shows a typical single-channel recording from an excised insideout patch of hippocampal neuron and the effect of low pH i on the patch current. The traces, from top to bottom, are consecutive segments obtained at a patch membrane potential of 0 mV. Pipette solution was 300 NaC1 (pH 7.2). The solution perfusing the cytoplasmic side of the membrane was 1200 NaC1 (pH 7.2) for the first two control traces (A), and 1200 NaC1 (pH 3.9) for the following traces (B). The control traces show an inward current which, given the zero voltage and the salt gradient conditions, indicates that the underlying channel is anion selective.
Single-channel conductance as derived from a current voltage plot (not shown) and channel kinetics indicate that this is the background C1- channel frequently seen and already reported in this preparation [36]. When the internal 1200 mM NaC1 (pH 7.2) is replaced by the low pH i solution (1200 mM NaC1 (pH 3.9)), an inhibitory effect on the C1- current is observed (Fig. 1). This inhibition consists of a depression of single-channel activity. The channel tends to open more rarely, and openings are no longer in bursts but appear as shorter and sparser separate events. Current amplitude also decreases until C1- channel activity subsides completely. Soon after the above changes have been completed, occasional openings appear with current going in the opposite direction, that is, positive with respect to the base line. As patch conditions are unchanged (zero holding voltage, and 1200 mM NaC1 (pH 3.9) H300 mM NaC1 (pH 7.2)), the outward current observed must be carried either by Na + or H + ions. This current was not abolished upon returning to physiological pH conditions, indicating that the activation of the channel by low pH was irreversible. To study this current in more detail silent patches mv
2B
2A
mV
50
40
30
2O
10
m
-
• ....
: -
"
-
-20
A
-20
-30
-30
-40
-40
p A: ms:
Be. 26 178
-50
pA: ms:
95.81 145
Fig. 2. Single-channel currents of a cationic channel activated by low p H i and amplitude histograms obtained at different m e m b r a n e potentials and perfusing conditions. Pipette solution was 300 m M NaC1 (pH 7.2) and the solution perfusing the inside-out patch was 300 m M NaC1 (pH 4.1) (A), and 1200 m M NaC1 (pH 4.1) (B). Current records were obtained by the staircase stimulation protocol (see Methods). The corresponding amplitude histograms shown on the fight of each record were constructed by plotting on the ordinate the square root of the n u m b e r of samples in each bin (bin size 0.08 pA). Calibration is given in the figure.
226
SbmV pipette: .
.
.
-
.
300
NaCl
.
1200
NaCI
-8
Fig. 3. Current-voltage relations of the proton-activated cationic channel in two different ionic conditions. Data are from the experiment shown in Fig. 2. Pipette solution 300 mM NaCI (pH 7..2). Perfusing solutions 300 mM NaC1 (pH 4.1) (dots), and 1200 mM NaC1 (pH 4.1) (bars). Single-channel currents were estimated from the amplitude histograms. Each data point represents a separate measurement. The continuous lines were fitted to the data points by eye. In symmetrical 300 mM NaC1 (dots), slope conductance is approx. 110 pS. In salt gradient conditions (bars) the interpolated line predicts a reversal potential of - 34 mV.
with no o b s e r v e d c h a n n e l activity were s u b j e c t e d to low p H i. Fig. 2 illustrates a r e p r e s e n t a t i v e e x p e r i m e n t where the excised i n s i d e - o u t p a t c h is p e r f u s e d with 300 m M NaC1 ( p H 4.1) (A), a n d after r e p l a c i n g this solution with 1200 m M NaC1 ( p H 4.1) (B). Pipette s o l u t i o n in this e x p e r i m e n t was 300 m M NaC1 ( p H 7.2). W i t h s y m m e t r i c a l 300 m M NaC1, b u t h i g h H + gradient, the reversal potential, as shown b y single-channel r e c o r d s a n d a m p l i t u d e h i s t o g r a m s (see also Fig. 3), is n e a r zero, A
closed
300
NaCl,
as e x p e c t e d if N a ÷ a n d n o t H + ions were the charge carriers, y m m e t r i c a l 150 m M NaC1 solutions were occasionally used with the s a m e results. A s higher salt c o n c e n t r a t i o n p r o d u c e s larger currents, we have pred o m i n a n t l y u s e d these solutions). W h e n the s a m e p a t c h is s u b j e c t e d to NaC1 g r a d i e n t c o n d i t i o n s (1200 m M NaC1 ( p H 4.1) II 300 m M NaC1 ( p H 7.2)) the reversal p o t e n t i a l is shifted c o n s i d e r a b l y to negative voltages. A s can b e seen in Fig. 3 which shows the current-voltage
pH 7 . 2 / / 3 0 0 NaCl, pH 7.2
B
J,l, /6rrm 'HII-
pA :
20
pA : 20 ms : 1600 Fig. 4. Steady-state kinetics of the proton-activated cationic channel. (A) Single-channel recording in symmetrycal 300 mM NaC1 (pH 7.2). Traces are consecutive 1600-ms segments taken at holding potential of 40 inV. The patch was previously subjected to low pH i to activate the cationic channel, and then returned to physiological pH. (B) Amplitude histogram from a 8-s segment of continuous recording which includes the two records shown in (A). The histogram abscissa spans 20 pA, and represents also the current calibration (pA between the solid lines enclosing the single-channel records) for (A). The ordinate plots the square root of the number of samples in each 0.04 pA bin.
227 plot constructed with data from this experiment, the reversal potential in NaC1 gradient conditions is - 3 4 mV (the average from 11 patches gave a mean + S.D. of - 31 + 3 mV). This value is very close to the theoretical Nernst potential of - 3 2 mV for a membrane exclusively permeable to Na + (activity coefficients of 0.710 and 0.645 have been used, respectively, for 300 and 1200 mM NaC1 solutions). This result indicates that the channel is highly selective for Na + over H +. K + selectivity was also tested by substituting 300 mM KCI (pH 4.1) for 300 mM NaC1 (pH 4.1) on the intracellular side of the patch. In three patches so tested the reversal potential was found to shift 13, 14, and 18 mV respectively, indicating that K + is less permeant than Na ÷. The mean shift of 15 mV in the reversal potential would be accounted for with the Nernst equation by permeabilities of K ÷ relative to Na + (P~JPNa) of 0.69. Fig. 2 also shows that channel activity is a function of voltage: openings at positive voltages are greater than at negative voltages. Although a quantitative analysis of the voltage dependence of channel activity was not carried out, this apparent voltage sensitivity was seen in virtually all the experiments. In general, at negative voltages the channel opens rarely and many sweeps show no openings. As the patch is depolarized, the channels open more frequently, and double (and more) openings appear, reflecting an increase in open probability. Fig. 4 illustrates the kinetics of this cationic channel, by showing a segment of continuous recording taken at 40 mV in symmetrical 300 mM NaC1 (pH 7.2). The patch was initially subjected to 300 mM NaC1 (pH 4.2) solution to activate the cation channel (not shown), then returned to physiological pH. This figure shows that channel activation by low pH i is not a transient phenomenon, but more likely results from a permanent modification of the channel. An interesting feature of this channel is that for the recording frequency range used, the open-channel noise is not significantly different from the dosed channel noise. The amplitude histogram of Fig. 4B made from a 8-s segment of continuous recording which includes the two blocks shown in Fig. 4A illustrates this point more convincingly as well as showing the absence of any subconductance state for this channel. The low open-channel noise, reflecting the little flickering of the channel, indicates the absence of short-lived dosed states. Discussion
This paper reports on a study at the single-channel level of a cationic current elicited by low pH i applied to membrane patches of rat hippocampal neurons. The channel has a conductance of approx. 110 pS in symmetrical 300 mM NaC1, selects very strongly for Na +
over CI-, and is active within a wide voltage range (from +75 mV to - 7 5 mV). The channel remains active even after physiological pH is restored. The cationic channel is not activated by low pH applied extracellularly. Several authors have reported H currents in conditions of low intracellular pH values. Mozhayeva and Naumov [28] suggested that in nerve fibers the H ÷ current permeates through Na + channels (also see, Ref. 30), whereas the presence of a specific H ÷ channel has been proposed by Thomas and Meech [29] in the membrane of snail neurons. Regardless of the type of channel that sustains the current, the cationic current we described in this paper cannot be identified as a H ÷ current for the following two reasons. The strongest point is that in conditions of high proton gradient (with expected reversal for H ÷ of approx. - 1 8 0 mV), but otherwise symmetrical salt concentration (300 mM NaC1 II 300 mM NaC1), the reversal potential is near zero (Figs. 2A and 3). This result, along with the nearly ideal Nernst reversal potential shift of - 31 mV for a salt gradient of 1200 mM NaCI II 300 mM NaC1 convincingly indicate that N a ÷ ions and not H ÷ ions are the current carriers. In addition, the H + currents described by other authors showed timedependent characteristics, inactivating in hundred of ms [30]. The cationic current we describe h e r e is instead active within a wide voltage range, and present in steady-state condition at all voltages tested (from + 75 mV to - 7 5 mV), indicating time-independent kinetics. The observation that the block of the C1- channel always preceded the appearance of the cationic channel (cf. Fig. 1) initially led us to consider the possibility of a transformation of the CI- channel into a cationic channel by low pH i. This appeared a possible interpretation as the background C1- channel has been shown to have a significant permeability to cations [36], which might be determined by bipolar structures in the channel pore ( - O H groups), or by charged amino acids which could be affected by pH. In addition, such a transformation of an ionic channel type into another upon pH manipulation has been reported by other authors. Konnerth et al. [31], for instance, have suggested that in chick dorsal root ganglion cells Ca 2÷ channels assume a distinct conformational state, selective to Na ÷, by lowering the internal pH. It is worth noting here that the most convincing evidence provided to support this view was the 'temporal exclusiveness' of the Ca 2÷ and Na ÷ currents, a feature we frequently observed in our experiments with the C1- and Na ÷ currents.' There are, however, at least two reasons why this mechanism is unlikely to be applicable here. The first is that cationic currents can be induced by low pH i in silent patches. The second is that in experimental protocols similar to that illustrated in Fig. 1, we noticed sometimes, on returning to physiological pH, the reap-
228 pearance of the C1- channel, which would superimpose to the still active cationic channel. Four observations need to be accounted for in considering a possible mechanism of activation of the cationic current by low p H i. (1) The high proton concentration (pH i lower than 4.5) required to activate the cationic current. (2) The inefficacy of low p H when applied extracellularly. (3) The delay in activation of the cationic channel. (4) The persistance of the cationic channel activity upon restoring physiological conditions. (1) Such a low value of p H could be expected to induce changes in parts of proteins extending from the cytoplasmic side of the membrane. Low p H is known to modify the state of several proteins found in solution [37]. Such an effeot is presumably involved in the p H block of the C1- channel seen in Fig. 1, and may also be responsible for the activation of the cationic channel. (2) The sideness of the pH effect also argues for an influence of p H upon the state of the channel through protonation of the exposed cytoplasmic portion of the protein. It seeems less likely that such proton binding sites lie within the permeation path (selectively filter) of the channel, as protons could presumably reach them from either side of the pore. Still it may be possible that protons can access such a pore-oriented activation sites, but are only accessible to the pore from the extracellular side when the channel is in the pore-open conformation. (3) pH-induced structural changes of proteins are relatively slow processes [37]. The 1-2 rain delay between application of the low p H i solution and activation of the cationic channel argues for a slow pH-induced modification of the channel protein. The finding that pH values higher than 4.5 do not cause activation of the cationic channel indicates that if removal of some steady state inactivation gate were responsible for activation of this channel, then such a gate is relatively resistant to pH-induced changes. It is interesting to notice that in this context the C1- channel is more susceptible to modification by p H as it is already blocked at pH values near 5.5 (Franciolini and Nonner, in preparation). This may also argue against the idea that C1- channels are being transformed into a cationic channel. (4) The persistance of the cationic channel upon returning to physiological p H (Fig. 4) shows that the activation of this channel type by low p H i is irreversible. This is what would be expected if the low pH was indeed removing or altering some steady-state inactivation gate responsible for keeping this channel closed. It is also interesting to notice that the C1- channel rarely recovers from the low p H i exposure, even after a long period of time in the p H 7.2 solution. This may also be due to denaturation of the channel protein by the low p H we used. Taken together these four points suggest that the
activation of the cationic channel is mediated by pH, probably via an unfolding of the channel protein, which releases it from a long-term inactivation state. The effects reported in this paper have been obtained in experimental conditions (excised patches and low pH values) far from those experienced by cells physiologically. These findings cannot therefore be extrapolated to intact neuronal cells. The experiments reported here were directed at biophysical investigation of the ion channel itself, and no physiological significance can be claimed, p H has traditionally been used to probe the functional parameters of ion channels and much useful information has been elucidated from these types of studies [3,6,7,10,18,26,27,31,37]. Through this approach and by other manipulations we hope to further define the relationship of structure to activity in this new channel type.
Acknowledgments I thank R. N u m a n n and A. Petris for helpful discussion in the preparation of the manuscript. This work was supported by grant Progetto Bilaterale No. 880051404/115 from the Italian Consiglio Nazionale Ricerche.
References 1 Tasaki, I., Singer, I. and Takenaka, T. (1965) J. Gen. Physiol. 48, 1095-1123. 2 Hille, B. (1968) J. Gen. Physiol. 51, 221-236. 3 WoodhuU,A.M. (1973) J. Gen. Physiol.61, 687-708. 4 Schauf, C.L. and Davis, F.A. (1976) J. Gen, Physiol.67, 185-195. 5 Carbone, E., Testa, P.L. and Wartke, E. (1981) Biophys. J. 35, 393-413. 6 Campbell, D.T. (1982) Nature 298, 165-167. 7 Begenisich,T. and Danko, M. (1983)J. Gen. Physiol. 82, 599-618. 8 Shrager, P. (1974) J. Gen. Physiol. 64, 666-690. 9 Hagiwara, S., Miyazaki, S., Moody, W. and Patlak, J. (1978) J. Physiol. 279, 167-185. 10 Wanke, E., Carbone, E. and Testa, P.L. (1979) Biophys. J. 26, 319-324. 11 Blatz, A.L. (1980) Fed. Proc. 39, 2073. 12 Moody, Jr., W. and Hagiwara, S. (1982) J. Gen. Physiol. 79, 115-130. 13 Cook, D.L., Ikeuchi, M. and Fujimoto, W.Y. (1984) Nature 311, 269-271. 14 Chesnais, J.M., Coraboeuf, E., Sauviat, M.P. and Vassas, J.M. (1975) J. Mol. Cell Cardiol. 6, 627-642. 15 Kohlhardt, M,, Happ, K. and Figulla, H.R. (1976) PfluegersArch. 366, 31-38. 16 Vogel,S. and Sperelakis,N. (1977) Am. J. Physiol.233, C99-C103. 17 Yatani, A. and Goto, M. (1983) Jpn. J. Physiol. 33, 403-415. 18 Iijima, T.S., Ciani, S. and Hagiwara, S. (1986) Proc. Natl. Acad. Sci. USA 83, 654-658. 19 Prod'horn, B., Pietrobon, D. and Hess, P. (1987) Nature 329, 243-246. 20 Krafte, D.S. and Kass, R.S. (1988) J. Gen. Physiol. 91, 641-657. 21 Hurter, O.H. and Warner, A.E. (1967) J. Physiol. 189, 403-425. 22 Woodbury, J.W. and Miles, P.R. (1973) J. Gen. Physiol. 62, 324-353.
229 23 Loo, D.F., McLarnon, J.G. and Vaughan, P. (1981) Can. J. Physiol. Pharmacol. 59, 7-13. 24 Blatz, A.L. and Magleby, K.L. (1985) Biophys. J. 47, 119-123. 25 Landau, E.M., Gavish, B., Nachshen, D.A. and Lotan, I. (1981) J. Gen. Physiol. 77, 647-666. 26 Brodwick, M.S. and Eaton, D.C. (1978) Science 200, 1494-1496. 27 Nonner, W., Spalding, B.C. and Hille, B. (1980) Nature 284, 360-363. 28 Mozhayeva, G.N. and Naumov, A.P. (1983) Pfluegers Arch. 396, 163-173. 29 Thomas, R.C. and Meech, R.W. (1982) Nature 299, 826-828. 30 Byerly, L., Meech, R. and Moody, Jr., W. (1984) J. Physiol. 351, 199-216. 31 Konnerth, A., Lux, H.D. and Morad, M. (1987) J. Physiol. 386, 603-633.
32 Davies, N.W., Lux, H.D. and Morad, M. (1988) J. Physiol. 400, 159-187. 33 Hanke, W. and Miller, C. (1983) J. Gen. Physiol. 82, 25-45. 34 Kaufman, L.M. and Barrett, J.N. (1983) Science 220, 1394-1396. 35 Hamill, O.P., Marry, A., Neher, E., Sakmann, B. and Sigworth, F.J. (1981) Pfluegers Arch. 391, 85-100. 36 Franciolini, F. and Nonner, W. (1987) J. Gen. Physiol. 90, 453-478. 37 Privalov, P.L. (1979) Stability of proteins, in Advances of Protein Chemistry, Vol. 33 (Anfinsen, C.B., Edsall, J.T. and Richards, F.M., eds), pp. 167-241, Academic Press, New York. 38 Ehrestein, G. and Fishman, H.M. (1971) Nature New Biol. 233, 16-17.
223
Elsevier BBAMEM 74798
Single-channel currents activated by low intracellular pH in cultured hippocampal neurons of rat Fabio Franciolini Istituto Biologia CeUulare UnioersiMdi Perugia, Perugia (Italy) and Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL (U.S.A.)
(Received 11 July 1989) (Revised manuscript received1 November 1989)
Key words: Single-channelcurrent; Patch damp; Ion channel; pH; (Rat neuron)
Patch damp technique was applied to the plasma membrane of cultured hippucampai neurons of rat. Elementary currents of a cation-selective channel were elicited by low intraceilular pH (pH i 3.5-4.5). Channel activity starts with 1-2 min delay from the application of low pH i, and persists upon restoration of physiological pH conditions. The channel has a conductance of approx. 110 pS in symmetrical 300 mM NaCI, and is strongly selective for cations over anions. The channel is active over the whole voltage range tested (from + 75 mV to - 7 5 mV). Mean open time is function of voltage, increasing with depolarization. Low pH applied extracellularly did not activate the channel.
Introduction The effects of high proton concentration on ionic channel activity have been extensively studied. Reports broadly fall into three categories. (1) Inhibition of channel activity. Low (external or internal) p H has been shown to block conduction in the squid giant axon [1], and to decrease conductance or activity of Na ÷ channels [2-7], K ÷ (delayed rectifier, inward rectifier, Ca2+-activated) channels [4,8-13], Ca 2÷ channels [14-20], C1- channels [21-24], and acetylcholine receptor [25]. A few studies have also been done on the effect of intracellular p H on the inactivation of the Na ÷ channel. Two apparently conflicting reports are mentioned here. Na + current inactivation is removed or greatly reduced by high p H i in squid giant axon [26], but by low p H i in frog skeletal muscle [27]. (2) Production of H ÷ currents. H ÷ currents have been recently described by several authors in different preparations. Mozhayeva and Naumov [28] report a H ÷ current at p H lower than 4 that permeates through Na ÷ channels. In snail neurons, H + currents have been first reported by Thomas and Meech [29], and described in more detail by Byerly et al. [30]. The latter paper shows that H + currents go through specific channels which are very selective for H +.
Correspondence: F. Franciolini, Istituto BiologiaCeUulare,via Pascoli 1, 06100 Perugia, Italy.
(3) Transformation of one channel type into another. Lux and co-workers [31,32] found that low external p H (pHo) activates a transient N a ÷ current. Two major observations induced the authors to conclude that the Na ÷ current activated by low p H o flows through a transformed state of a Ca 2÷ channel. First, the voltagegated Ca 2÷ current disappeared rapidly during the activation of proton-induced Na ÷ current. Ca 2÷ current reappeared, however, while Na + current inactivated, upon restoration of physiological p H conditions. This 'temporal exclusiveness' of the two currents suggested that both Na + and Ca 2+ currents use the same Ca 2+ channel in two selectively distinct states. Additional evidence in support of this view was the finding that the proton-activated Na + current was blocked by organic (diltiazem, verapamil) and inorganic (Ni and Cd) Ca 2÷ channel blockers at concentrations similar to those required to block the Ca 2÷ currents. For completeness, one report has also appeared on proton modulation of C1- channels reconstituted from Torpedo electroplax [33], which shows that lowering pH increases the open probability of the channel, whereas rising it has the opposite effect. A change in p K of certain titratable groups upon opening of the channel is proposed to explain the pH-modulation of the channel gating. This paper reports on the activation of a cationic channel by low internal pH. The results are discussed in the light of previous reports, to try to determine to which category the effects reported can be ascribed, and
0005-2736/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
224 what possible mechanisms underly the proton-activated cationic channel in cultured hippocampal neurons of rat.
maintaining that value for tens of seconds to minutes, or by stimulating the membrane with a staircase pattern of voltage steps that started with positive steps to activate the channels, and applied over a period of time during which sufficient activity was sustained (for details, cf. Ref. 36). Current amplitude histograms of single-channel recordings were constructed for both stimulation protocols, and used to measure single-channel current and channel open probability. Single-channel current was measured as interpeak distance, and channel open probability calculated from the integral areas of the amplitude histogram peaks. The solutions used contained either 300 or 1200 mM of bulk salt (NaC1 or KC1), 1 mM EGTA, and 2 mM of pH buffer. Solutions where the pH was adjusted to 7.2 were buffered with Mops, and those adjusted to pH 3.5-4.5 were buffered with citric acid. In the text, these solutions are referred to by specifying the concentration and species of the bulk salt, and the pH; for instance, 300 NaC1 (pH 4.2). Effective change of test solutions at the cytoplasmic side of the patch in the inside-out configuration was obtained with a perfusion system similar to that described by Franciolini and Nonner [36]. Experiments were carried out at 25-28 o C.
Methods
Experiments were carried out on hippocampal neurons dissected from 18-day-old rat embryos, and plated on collagene/polylysine-coated culture dishes containing basic tissue culture medium (N5) supplemented with a fraction of horse serum [34]. Neurons were kept in culture at 34°C in an atmosphere containing 5% CO 2 for 1-3 weeks before the experiment. The patch clamp method [35] was used to record single-channel currents in the inside-out configuration, that is, with the cytoplasmic side of the membrane exposed to the perfusing solution. A homemade patch clamp amplifier was used for voltage clamping and current amplification. Single-channel currents were filtered at 0.5 kHz ( - 3dB), digitized at 400 #s sampling rate, and analyzed with a laboratory computer system (Nova 4, Data General). Single-channel records were taken in either steady-state conditions attained by stepping the membrane voltage to the desired level, and A
12OO NaCI,
pH Z 2 / / 3 0 0
NaCI,
pH 7.2
closed
B
12OO N a C I ,
pH 3 . 9 / / 3 0 0
NaCI, pH 7.2
/
~| ..I
LLI.t_.I _h
_]_|j~
pA : ms :
25 1560
Fig. 1. Activation of the cationic channel by low pH i. Single-channel recordings from an excised inside-out patch of hippocampal neuron of rat were taken in steady-state conditions at holding potential of 0 mV. The traces are consecutive 1560-ms segments interrupted by 20-ms blank intervals due to data aquisition. Pipette solution was 300 m M NaC1 (pH 7.2) and perfusing solution was 1200 m M NaC1 (pH 7.2) for the first two control traces (A), and 1200 m M NaC1 (pH 3.9) for the following traces (B). The traces shown in (B) were taken 80 s after solution change. Calibration: solid lines enclosing single channel recordings span 25 pA.
225 Results
In an attempt to elucidate the permeation mechanism of the background C1- channel in hippocampal neurons of rat, a series of experiments were carried out at low intracellular pH, ranging from 7.0 to 4.0. Low pH i was found to abolish the activity of the background C1- channel (Franciolini and Nonner, unpublished data). Often, however, the activation of a single-channel cationic current with distinctive conductance and kinetics was observed, concurrent with the depression of C1current. This is illustrated in Fig. 1 which shows a typical single-channel recording from an excised insideout patch of hippocampal neuron and the effect of low pH i on the patch current. The traces, from top to bottom, are consecutive segments obtained at a patch membrane potential of 0 mV. Pipette solution was 300 NaC1 (pH 7.2). The solution perfusing the cytoplasmic side of the membrane was 1200 NaC1 (pH 7.2) for the first two control traces (A), and 1200 NaC1 (pH 3.9) for the following traces (B). The control traces show an inward current which, given the zero voltage and the salt gradient conditions, indicates that the underlying channel is anion selective.
Single-channel conductance as derived from a current voltage plot (not shown) and channel kinetics indicate that this is the background C1- channel frequently seen and already reported in this preparation [36]. When the internal 1200 mM NaC1 (pH 7.2) is replaced by the low pH i solution (1200 mM NaC1 (pH 3.9)), an inhibitory effect on the C1- current is observed (Fig. 1). This inhibition consists of a depression of single-channel activity. The channel tends to open more rarely, and openings are no longer in bursts but appear as shorter and sparser separate events. Current amplitude also decreases until C1- channel activity subsides completely. Soon after the above changes have been completed, occasional openings appear with current going in the opposite direction, that is, positive with respect to the base line. As patch conditions are unchanged (zero holding voltage, and 1200 mM NaC1 (pH 3.9) H300 mM NaC1 (pH 7.2)), the outward current observed must be carried either by Na + or H + ions. This current was not abolished upon returning to physiological pH conditions, indicating that the activation of the channel by low pH was irreversible. To study this current in more detail silent patches mv
2B
2A
mV
50
40
30
2O
10
m
-
• ....
: -
"
-
-20
A
-20
-30
-30
-40
-40
p A: ms:
Be. 26 178
-50
pA: ms:
95.81 145
Fig. 2. Single-channel currents of a cationic channel activated by low p H i and amplitude histograms obtained at different m e m b r a n e potentials and perfusing conditions. Pipette solution was 300 m M NaC1 (pH 7.2) and the solution perfusing the inside-out patch was 300 m M NaC1 (pH 4.1) (A), and 1200 m M NaC1 (pH 4.1) (B). Current records were obtained by the staircase stimulation protocol (see Methods). The corresponding amplitude histograms shown on the fight of each record were constructed by plotting on the ordinate the square root of the n u m b e r of samples in each bin (bin size 0.08 pA). Calibration is given in the figure.
226
SbmV pipette: .
.
.
-
.
300
NaCl
.
1200
NaCI
-8
Fig. 3. Current-voltage relations of the proton-activated cationic channel in two different ionic conditions. Data are from the experiment shown in Fig. 2. Pipette solution 300 mM NaCI (pH 7..2). Perfusing solutions 300 mM NaC1 (pH 4.1) (dots), and 1200 mM NaC1 (pH 4.1) (bars). Single-channel currents were estimated from the amplitude histograms. Each data point represents a separate measurement. The continuous lines were fitted to the data points by eye. In symmetrical 300 mM NaC1 (dots), slope conductance is approx. 110 pS. In salt gradient conditions (bars) the interpolated line predicts a reversal potential of - 34 mV.
with no o b s e r v e d c h a n n e l activity were s u b j e c t e d to low p H i. Fig. 2 illustrates a r e p r e s e n t a t i v e e x p e r i m e n t where the excised i n s i d e - o u t p a t c h is p e r f u s e d with 300 m M NaC1 ( p H 4.1) (A), a n d after r e p l a c i n g this solution with 1200 m M NaC1 ( p H 4.1) (B). Pipette s o l u t i o n in this e x p e r i m e n t was 300 m M NaC1 ( p H 7.2). W i t h s y m m e t r i c a l 300 m M NaC1, b u t h i g h H + gradient, the reversal potential, as shown b y single-channel r e c o r d s a n d a m p l i t u d e h i s t o g r a m s (see also Fig. 3), is n e a r zero, A
closed
300
NaCl,
as e x p e c t e d if N a ÷ a n d n o t H + ions were the charge carriers, y m m e t r i c a l 150 m M NaC1 solutions were occasionally used with the s a m e results. A s higher salt c o n c e n t r a t i o n p r o d u c e s larger currents, we have pred o m i n a n t l y u s e d these solutions). W h e n the s a m e p a t c h is s u b j e c t e d to NaC1 g r a d i e n t c o n d i t i o n s (1200 m M NaC1 ( p H 4.1) II 300 m M NaC1 ( p H 7.2)) the reversal p o t e n t i a l is shifted c o n s i d e r a b l y to negative voltages. A s can b e seen in Fig. 3 which shows the current-voltage
pH 7 . 2 / / 3 0 0 NaCl, pH 7.2
B
J,l, /6rrm 'HII-
pA :
20
pA : 20 ms : 1600 Fig. 4. Steady-state kinetics of the proton-activated cationic channel. (A) Single-channel recording in symmetrycal 300 mM NaC1 (pH 7.2). Traces are consecutive 1600-ms segments taken at holding potential of 40 inV. The patch was previously subjected to low pH i to activate the cationic channel, and then returned to physiological pH. (B) Amplitude histogram from a 8-s segment of continuous recording which includes the two records shown in (A). The histogram abscissa spans 20 pA, and represents also the current calibration (pA between the solid lines enclosing the single-channel records) for (A). The ordinate plots the square root of the number of samples in each 0.04 pA bin.
227 plot constructed with data from this experiment, the reversal potential in NaC1 gradient conditions is - 3 4 mV (the average from 11 patches gave a mean + S.D. of - 31 + 3 mV). This value is very close to the theoretical Nernst potential of - 3 2 mV for a membrane exclusively permeable to Na + (activity coefficients of 0.710 and 0.645 have been used, respectively, for 300 and 1200 mM NaC1 solutions). This result indicates that the channel is highly selective for Na + over H +. K + selectivity was also tested by substituting 300 mM KCI (pH 4.1) for 300 mM NaC1 (pH 4.1) on the intracellular side of the patch. In three patches so tested the reversal potential was found to shift 13, 14, and 18 mV respectively, indicating that K + is less permeant than Na ÷. The mean shift of 15 mV in the reversal potential would be accounted for with the Nernst equation by permeabilities of K ÷ relative to Na + (P~JPNa) of 0.69. Fig. 2 also shows that channel activity is a function of voltage: openings at positive voltages are greater than at negative voltages. Although a quantitative analysis of the voltage dependence of channel activity was not carried out, this apparent voltage sensitivity was seen in virtually all the experiments. In general, at negative voltages the channel opens rarely and many sweeps show no openings. As the patch is depolarized, the channels open more frequently, and double (and more) openings appear, reflecting an increase in open probability. Fig. 4 illustrates the kinetics of this cationic channel, by showing a segment of continuous recording taken at 40 mV in symmetrical 300 mM NaC1 (pH 7.2). The patch was initially subjected to 300 mM NaC1 (pH 4.2) solution to activate the cation channel (not shown), then returned to physiological pH. This figure shows that channel activation by low pH i is not a transient phenomenon, but more likely results from a permanent modification of the channel. An interesting feature of this channel is that for the recording frequency range used, the open-channel noise is not significantly different from the dosed channel noise. The amplitude histogram of Fig. 4B made from a 8-s segment of continuous recording which includes the two blocks shown in Fig. 4A illustrates this point more convincingly as well as showing the absence of any subconductance state for this channel. The low open-channel noise, reflecting the little flickering of the channel, indicates the absence of short-lived dosed states. Discussion
This paper reports on a study at the single-channel level of a cationic current elicited by low pH i applied to membrane patches of rat hippocampal neurons. The channel has a conductance of approx. 110 pS in symmetrical 300 mM NaC1, selects very strongly for Na +
over CI-, and is active within a wide voltage range (from +75 mV to - 7 5 mV). The channel remains active even after physiological pH is restored. The cationic channel is not activated by low pH applied extracellularly. Several authors have reported H currents in conditions of low intracellular pH values. Mozhayeva and Naumov [28] suggested that in nerve fibers the H ÷ current permeates through Na + channels (also see, Ref. 30), whereas the presence of a specific H ÷ channel has been proposed by Thomas and Meech [29] in the membrane of snail neurons. Regardless of the type of channel that sustains the current, the cationic current we described in this paper cannot be identified as a H ÷ current for the following two reasons. The strongest point is that in conditions of high proton gradient (with expected reversal for H ÷ of approx. - 1 8 0 mV), but otherwise symmetrical salt concentration (300 mM NaC1 II 300 mM NaC1), the reversal potential is near zero (Figs. 2A and 3). This result, along with the nearly ideal Nernst reversal potential shift of - 31 mV for a salt gradient of 1200 mM NaCI II 300 mM NaC1 convincingly indicate that N a ÷ ions and not H ÷ ions are the current carriers. In addition, the H + currents described by other authors showed timedependent characteristics, inactivating in hundred of ms [30]. The cationic current we describe h e r e is instead active within a wide voltage range, and present in steady-state condition at all voltages tested (from + 75 mV to - 7 5 mV), indicating time-independent kinetics. The observation that the block of the C1- channel always preceded the appearance of the cationic channel (cf. Fig. 1) initially led us to consider the possibility of a transformation of the CI- channel into a cationic channel by low pH i. This appeared a possible interpretation as the background C1- channel has been shown to have a significant permeability to cations [36], which might be determined by bipolar structures in the channel pore ( - O H groups), or by charged amino acids which could be affected by pH. In addition, such a transformation of an ionic channel type into another upon pH manipulation has been reported by other authors. Konnerth et al. [31], for instance, have suggested that in chick dorsal root ganglion cells Ca 2÷ channels assume a distinct conformational state, selective to Na ÷, by lowering the internal pH. It is worth noting here that the most convincing evidence provided to support this view was the 'temporal exclusiveness' of the Ca 2÷ and Na ÷ currents, a feature we frequently observed in our experiments with the C1- and Na ÷ currents.' There are, however, at least two reasons why this mechanism is unlikely to be applicable here. The first is that cationic currents can be induced by low pH i in silent patches. The second is that in experimental protocols similar to that illustrated in Fig. 1, we noticed sometimes, on returning to physiological pH, the reap-
228 pearance of the C1- channel, which would superimpose to the still active cationic channel. Four observations need to be accounted for in considering a possible mechanism of activation of the cationic current by low p H i. (1) The high proton concentration (pH i lower than 4.5) required to activate the cationic current. (2) The inefficacy of low p H when applied extracellularly. (3) The delay in activation of the cationic channel. (4) The persistance of the cationic channel activity upon restoring physiological conditions. (1) Such a low value of p H could be expected to induce changes in parts of proteins extending from the cytoplasmic side of the membrane. Low p H is known to modify the state of several proteins found in solution [37]. Such an effeot is presumably involved in the p H block of the C1- channel seen in Fig. 1, and may also be responsible for the activation of the cationic channel. (2) The sideness of the pH effect also argues for an influence of p H upon the state of the channel through protonation of the exposed cytoplasmic portion of the protein. It seeems less likely that such proton binding sites lie within the permeation path (selectively filter) of the channel, as protons could presumably reach them from either side of the pore. Still it may be possible that protons can access such a pore-oriented activation sites, but are only accessible to the pore from the extracellular side when the channel is in the pore-open conformation. (3) pH-induced structural changes of proteins are relatively slow processes [37]. The 1-2 rain delay between application of the low p H i solution and activation of the cationic channel argues for a slow pH-induced modification of the channel protein. The finding that pH values higher than 4.5 do not cause activation of the cationic channel indicates that if removal of some steady state inactivation gate were responsible for activation of this channel, then such a gate is relatively resistant to pH-induced changes. It is interesting to notice that in this context the C1- channel is more susceptible to modification by p H as it is already blocked at pH values near 5.5 (Franciolini and Nonner, in preparation). This may also argue against the idea that C1- channels are being transformed into a cationic channel. (4) The persistance of the cationic channel upon returning to physiological p H (Fig. 4) shows that the activation of this channel type by low p H i is irreversible. This is what would be expected if the low pH was indeed removing or altering some steady-state inactivation gate responsible for keeping this channel closed. It is also interesting to notice that the C1- channel rarely recovers from the low p H i exposure, even after a long period of time in the p H 7.2 solution. This may also be due to denaturation of the channel protein by the low p H we used. Taken together these four points suggest that the
activation of the cationic channel is mediated by pH, probably via an unfolding of the channel protein, which releases it from a long-term inactivation state. The effects reported in this paper have been obtained in experimental conditions (excised patches and low pH values) far from those experienced by cells physiologically. These findings cannot therefore be extrapolated to intact neuronal cells. The experiments reported here were directed at biophysical investigation of the ion channel itself, and no physiological significance can be claimed, p H has traditionally been used to probe the functional parameters of ion channels and much useful information has been elucidated from these types of studies [3,6,7,10,18,26,27,31,37]. Through this approach and by other manipulations we hope to further define the relationship of structure to activity in this new channel type.
Acknowledgments I thank R. N u m a n n and A. Petris for helpful discussion in the preparation of the manuscript. This work was supported by grant Progetto Bilaterale No. 880051404/115 from the Italian Consiglio Nazionale Ricerche.
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